1). Splenocytes and liver infiltrating MNCs were isolated as described and resuspended in staining buffer consisting of 0.2% bovine serum albumin (BSA), 0.04% ethylenediaminetetraacetic acid (EDTA), and 0.05% sodium azide in phosphate-buffered saline (PBS). The cells were dispensed into
25-μL aliquots and incubated with antimouse Fc receptor blocking reagent (eBioscience, San Diego, CA) for 15 minutes at 4°C. Cells were washed and stained for 30 minutes at 4°C with cocktails containing combinations of fluorochrome conjugated monoclonal antibodies for the cell surface markers CD4, CD8a, CD44, CD62L, NK1.1, TCR Vα2, TCR Vβ5.1, 5.2 (Biolegend, San Diego, CA), and TCR-β (eBioscience). After staining, the cells were washed once with
PBS containing 0.2% BSA. For Ku 0059436 intracellular cytokine staining, splenic MNCs from dnTGFβRII, OT-I/dnTGFβRII/Rag1−/−, and OT-I/Rag1−/− mice were resuspended in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (Gibco-Invitrogen, Grand Island, NY), 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.5 μg/mL each of anti-CD3 (Biolegend) and anti-CD28 (Biolegend) or 10 μg/mL the OVA amino acid 257-264 peptide (GenScript, Piscataway, NJ). The cells were incubated at 37°C in a humidified 5% CO2 incubator. Brefeldin A (1 μg/mL) (Sigma-Aldrich, St. Louis, MO) was added after 1 hour incubation. The cells were RGFP966 chemical structure then incubated for 4 hours. The cells were stained for surface CD8a, NK1.1, and TCRβ, fixed, and permeabilized with BD Cytofix/Cytoperm Solution (BD Biosciences), then stained for intracellular IFNγ (BioLegend). Normal IgG isotype controls were used in parallel. A FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA) upgraded for the detection of five colors by Cytek Development (Fremont, CA) was used to acquire data, which were analyzed with Cellquest PRO software (BD Immunocytometry Systems). The liver from sacrificed mice were fixed in
4% paraformaldehyde, embedded in paraffin, cut into 4-μm sections, deparaffinized, stained with hematoxylin and eosin (H&E), and evaluated using light microscopy. Portal inflammation were evaluated by a “blinded” pathologist selleck compound using the following scoring system we have previously defined: 0, no inflammation; 1, minimal inflammation; 2, mild inflammation; 3, moderate inflammation; and 4, severe inflammation. Bile duct damage was graded as: 0, no significant changes; 1, mild change; 2, moderate to severe bile duct damage or bile duct loss. These data were expressed as the mean ± standard deviation (SD) and were evaluated with a two-tailed unpaired Mann-Whitney test, one-way analysis of variance (ANOVA) followed by a Bonferroni multiple comparison test, or a Kruskal-Wallis test followed by Dunn’s multiple comparisons test, as appropriate. We confirmed the composition of the splenic T-cell compartment in OT-I/dnTGFβRII/Rag1−/− and OT-II/dnTGFβRII/Rag1−/− mice.